Parameters of the labeling of mitogen-activated murine lymphocytes by [35S]methionine for two-dimensional gel electrophoresis I. Effect of culture conditions

John R. Kettman, Lotte Kuhn, Patricia Young, Ivan Lefkovits

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Labeling with [35S]methionine at a high specific activity is essential to the facile preparation of 2-dimensional gel electrophoretograms with the analytical 2-dimensional charge-size separation procedure (Anderson's ISODALT system). Mitogen-activated T and B lymphocytes subjected to low methionine concentrations would not proceed through cell cycle. In the case of activated B lymphocytes, the use of fetal bovine serum (FBS), dialyzed to lower endogenous methionine concentrations, prevented B cell growth even in the presence of otherwise satisfactory levels of methionine. High concentrations of [35S]methionine (> 300 mCi/l) induced B cell death, apparently by radiation damage. Despite these problems, good radioautograms and radiofluorograms of 2D electrophoretograms could be prepared by labeling activated B or T cells in bulk (106 cells/ml) with high specific activity [35S]methionine. The polypeptides labeled may be a biased sample since lymphoid cells do not proceed through cell cycle under these conditions. Small numbers (103) of activated T cells also yielded satisfactory samples but labeling of small numbers of activated B cells was not possible.

Original languageEnglish (US)
Pages (from-to)53-64
Number of pages12
JournalJournal of Immunological Methods
Volume88
Issue number1
DOIs
StatePublished - Apr 3 1986

Keywords

  • Autoradiography
  • Cell culture
  • Gel electrophoresis
  • Lymphocytes
  • Mitogens
  • Proteins
  • [S]methionine

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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